We propose to continue our research program to study and exploit intein-mediated protein splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, becomes excised from a host protein, the extein, in an autocatalytic manner. In protein trans-splicing the intein is split into two pieces ad splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all three phylogenetic domains, including several pathogens such as Mycobacterium tuberculosis (Mtb) where their splicing activity is required for DNA replication. Work from many laboratories, including our own, has painted a picture of protein splicing as being a slow, often inefficient, process taking several hours to reach completion. Very recent work has, however, caused us to drastically re-evaluate this dogma. This is because of a remarkable split intein from the cyanobacterium, Nostoc punctiforme (Npu intein) that supports protein splicing orders of magnitude faster than anything seen before (half-life of less than a minute). This finding raises several fascinating questions that form the core of this renewal application. First and foremost, how does this intein splice so fast? Are there other uncharacterized split inteins that splice as fast or even faster? Do these split proteins retain an structure prior to association and what is the mechanism of fragment complementation? How can we harness the ultra-fast kinetics of Npu (and other family members) for new applications? We will take a multi-pronged approach to study catalysis (Aim 1) and folding (Aim 2) of the Npu intein, as well as other as yet uncharacterized split inteins. For this we will employ the tools of enzymology, biophysics, bioinformatics and structural biology. All of these studies will be aided by our ability to incorporate a broad range of biophysical and biochemical probes (unnatural amino acids) into the proteins using chemical synthesis. In Aim 3, new technologies will be developed that exploit the properties of these new split inteins for both in vitro and in vivo protein chemistry applications. This includes developing functionally orthogonal split inteins, conditional split inteins and split inteins for semi-synthesis in cells. While expected to be broady applicable, these tools will be applied to a specific set of problems in chromatin biology of ongoing interest to my laboratory.